Methods, systems, and compositions for calpain inhabition

ABSTRACT

Methods, systems and compositions comprising novel peptidomimetics are disclosed that can be used to inhibit calpain and, more specifically, to treat tissue damage caused by pathologic activation of calpains.

BACKGROUND

Stroke by blockage of cerebral artery supplying blood to the brain is amajor cause of death and disability worldwide. Stroke in mammals,including in humans, often presents clinically as a disruption of bloodflow to the brain and other parts of the neurological system.Mechanistically, this disruption of blood flow often results inischemia, which can produce damage, death, and/or other injury to thetissue or organ with comprised blood flow. The etiology of ischemicinjury is sometimes mediated at the molecular level.

Despite its public heath significance, treatment of ischemic stroke islimited to thrombolysis by tissue plasminogen activator administeredintravenously within three hours of symptom onset. Only a smallpercentage (<5%) of stroke patients ultimately receive this treatment.

Calpain is a cysteine protease located in the cytosol of all cells,including neurons, and is implicated in cell death as part of thecalcium modulated cascade of enzymatic events after ischemic damage.Dysregulation of calpain, a prominent cysteine proteinase recognized toplay an important role in signal transduction, cell migration andregulation of apoptosis, has been implicated in a variety ofneurodegenerative disorders, including tissue damage, following stroke,traumatic brain and spine injury. It has also been linked topathogenesis of a variety of neurological diseases, such as multiplesclerosis, and Alzheimer's, Huntington's and Parkinson's disease. Thesedisorders are characterized by intracellular calcium overload leading toexcessive activation of calpains. In vivo inhibition of calpain usingcysteine protease inhibitors (e.g. peptide aldehydes, alpha-keto estersand amides) diminishes the extent of neuronal damage following ischemia;however, none of these inhibitors is specific for calpain, since theyalso effectively inhibit other cysteine proteases as well.

Treatments for neurodegenerative diseases and brain pathologies remainlimited. Previous calpain inhibitors have been studied as a potentialtreatment strategy for stroke, but these inhibitors are not selectivefor calpain and may incur unwanted side effects by their interactionwith other proteases. Thus, an unmet need remains for methods, systems,and compositions for selectively inhibition of calpain and withoutunwanted side effects.

BRIEF SUMMARY

Without limitation to only those embodiments expressly disclosed hereinand without disclaiming any embodiments, some embodiments of theinvention comprise methods, systems, and compositions to selectivelyinhibit biologically active calpain. In some embodiments, suchinhibition may be used to treat cell, tissue, or organ damage in apatient by administering to said patient a therapeutically effectiveamount of a peptidomimetic specific for calpain.

BRIEF DESCRIPTION OF THE DRAWINGS

Some embodiments of the present invention will now be described, by wayof example only and without disclaimer of other embodiments, withreference to the accompanying drawings (in which “B27-HYD” refers to SEQID NO: 5, as discussed herein):

FIG. 1 is a chart showing infarct volume in treated and controlsubjects.

FIG. 2 shows the results of neurological testing in control and treatedsubjects.

FIG. 3 is a photograph of stained tissue sections for control andtreated brain tissue.

FIG. 4 shows data concerning relative αII-spectrin levels in test andcontrol animals.

FIG. 5 shows data indicating that SEQ ID NO: 5 reduces rat serum albumin(RSA) leakage into the ischemic brain parenchyma.

FIG. 6 shows data indicating that SEQ ID NO: 5 treatment decreasesfibrin deposition outside the cerebral microvasculature.

FIG. 7 shows data indicating that SEQ ID NO: 5 modulates eNOSphosphorylation state.

FIG. 8 shows data indicating that post-injury treatment with SEQ ID NO:5 induces eNOS-Thr495 dephosphorylation

DETAILED DESCRIPTION

Without limitation to only those embodiments expressly disclosed hereinand without disclaiming any embodiments, some embodiments of theinvention comprise methods, systems, and compositions to selectivelyinhibit biologically active calpain. In some embodiments, suchinhibition may be used to treat cell, tissue, or organ damage in apatient by administering to said patient a therapeutically effectiveamount of a peptidomimetic specific for calpain.

We have discovered, isolated, and/or made novel structures for selectivecalpain inhibitors which, among other uses, can reduce the effects ofcerebral ischemia in mammals, including without limitation, humanbeings. Without limitation, some embodiments of the present inventionare directed to methods, systems, and/or compositions for reducingtissue damage in a mammal resulting from excessive activation ofcalpain. Such compositions useful for this purpose comprisecalpastatin-based peptidomimetics which are specific inhibitors ofcalpain.

Some embodiments comprise peptidomimetic versions of compounds thatcontain a number of non-peptidic chemical bonds, non-proteinogenic aminoacids, and/or conformational constraints.

We have isolated, synthesized and/or modified a nucleotide as well asrelated amino acid sequence of the native 27-amino acidcalpastatin-derived peptide to develop peptidomimetic calpain inhibitorsof some embodiments, as follows:

(SEQ ID NO: 1) acc caa tgg cta cta cct acc taa gag gaa ttg ggt aaa aga gaa gtc aca att cct cca aaa tat agg gaa  cta ttg gct(SEQ ID NO: 2) Asp Pro Met Ser Ser Thr Tyr Ile Glu Glu Leu Gly Lys Arg Glu Val Thr Ile Pro Pro Lys Tyr Arg Glu  Leu Leu Ala

In some embodiments, side-chain to side-chain cyclization is performedbetween Lys²¹ and Glu²⁴ side chains of SEQ ID No. 2.

Some embodiments comprise the following natural amino acid replacementsin SEQ ID NO: 2:

-   Arg²³ to Val²³-   Glu²⁴ to Ala²⁴-   Ala²⁷ to Pro²⁷,

thus providing a sequence Asp Pro Met Ser Ser Thr Tyr Ile Glu Glu LeuGly Lys Arg Glu Val Thr Ile Pro Pro Lys Tyr Val Ala Leu Leu Pro (SEQ IDNO: 3). In some embodiments, the hydrophobic sequence Ala Val Leu LeuAla Leu Leu Ala Pro (SEQ ID NO: 4) is added to the C-terminus of SEQ IDNO: 3 to provide a sequence of Asp Pro Met Ser Ser Thr Tyr Ile Glu GluLeu Gly Lys Arg Glu Val Thr Ile Pro Pro Lys Tyr Val Ala Leu Leu Pro AlaVal Leu Leu Ala Leu Leu Ala Pro (SEQ ID No: 5).

In some embodiments, one or more of the more usual peptidic bondsbetween the following amino acid pairs of SEQ ID NO: 2, SEQ ID NO: 3, orSEQ ID NO. 5 are replaced with non-peptidic reduced amide bondsrendering the peptidomimetic resistant to cleavage by proteolyticenzymes in vivo, and consequently improving its pharmacokineticproperties:

-   Ser⁴-Ser⁵-   Tyr⁶-Ile⁷-   Lys¹³-Arg¹⁴-   Glu¹⁵-Val¹⁶-   Lys²¹-Tyr²²-   Tyr²²-Arg²³-   Arg²³-Glu²⁴.

Moreover, in some embodiments, one or more of the following natural toun-natural amino acid substitutions are made in SEQ ID NO: 2, SEQ ID No:3, or SEQ ID NO: 5:

-   Ile¹⁸→Nva¹⁸-   Arg²³-Orn²³-   providing sequences:

(SEQ ID NO: 6) Asp Pro Met Ser Ser Thr Tyr Ile Glu Glu Leu Gly Lys Arg Glu Val Thr Nva Pro Pro Lys Tyr Orn Glu  Leu Leu Ala,(SEQ ID NO: 7) Asp Pro Met Ser Ser Thr Tyr Ile Glu Glu Leu Gly Lys Arg Glu Val Thr Nva Pro Pro Lys Tyr Arg Glu  Leu Leu Ala,(SEQ ID NO: 8) Asp Pro Met Ser Ser Thr Tyr Ile Glu Glu Leu Gly Lys Arg Glu Val Thr Ile Pro Pro Lys Tyr Orn Glu  Leu Leu Ala;(SEQ ID NO: 9) Asp Pro Met Ser Ser Thr Tyr Ile Glu Glu Leu Gly Lys Arg Glu Val Thr Nva Pro Pro Lys Tyr Val Ala  Leu Leu Pro, or(SEQ ID No: 10) Asp Pro Met Ser Ser Thr Tyr Ile Glu Glu Leu Gly Lys Arg Glu Val Thr Nva Pro Pro Lys Tyr Val Ala Leu Leu Pro Ala Val Leu Leu Ala Leu Leu Ala Pro.

In some embodiments, without limitation, the compounds contain ahydrophobic blood-brain-barrier (“BBB”) or membrane-permeant amino acidsequence linked by a polyethylene glycol linker to the N-terminus of thepeptidomimetic inhibitor. Without limitation, one example of ahydrophobic membrane-permeant amino acid sequence, and relatednucleotide sequence, comprising embodiments of the invention as follows:

(SEQ ID NO: 11) gcc gcg gta gcg ctg ctc ccg gcg gtc ctg ctg gcc ttg ctg gcg ccc (SEQ ID NO: 12)Ala Ala Val Ala Leu Leu Pro Ala Val Leu Leu Ala Leu Leu Ala Pro.Non-peptidic BBB permeant structures such as taurine, cholesterol, anonionic amphiphilic diethylene glycol methyl moiety, etc. can also beoptionally attached to the inhibitor.

Dysregulation of calpain, a cysteine proteinase recognized to play animportant role in signal transduction, cell migration and regulation ofapoptosis, has been implicated in a variety of neurodegenerativedisorders, including tissue damage, following stroke, traumatic brainand spine injury. These disorders are characterized by intracellularcalcium overload leading to excessive activation of calpains. In vivoinhibition of calpain using cysteine protease inhibitors (e.g. peptidealdehydes, alpha-keto esters and amides) diminishes the extent ofneuronal damage following ischemia; however, the relative nonspecificityof these cysteine proteinase inhibitors precludes a definitive role forcalpain participation. Calpastatin is the only inhibitor that isspecific for calpain, and it is generally accepted that the interactionof calpastatin with calpain is the most relevant mechanism responsiblefor the regulation of Ca²⁺-induced proteolysis. The deduced primarystructure of calpastatin consists of a non-inhibitory L-domain and fourrepeating inhibitory domains, each having an independent inhibitoryactivity against calpain and thus constituting a functional unit (SeeEmori et al, “All four repeating domains of the endogenous inhibitor forcalcium-dependent protease independently retain inhibitory activity.Expression of the cDNA fragments in Escherichia coli”, J. Biol. Chem.263, 2364-2370).

We previously identified two “hot spots” within the calpastatin moleculein which side chains of the most critical residues of calpastatininteract with hydrophobic pockets in calpain. Mutation of any of the keyresidues in either hot spot results in loss of inhibitory activity. Ourwork indicates that different regions in the inhibitory domain ofcalpastatin interact with complementary sites in calpain and undergocalpain-induced conformational changes that are crucial to inhibitor'saffinity and selectivity. Our results indicate that specific inhibitionof calpain in vivo by administration of novel membrane-permeantcalpastatin peptide(s) reduces infarct volume and neurological deficitsby blocking post-ischemic proteolysis of vital brain proteins.

EXAMPLES

The following examples of some embodiments of the invention are providedwithout limiting the invention to only those embodiments describedherein and without disclaiming any embodiments.

Example 1

Dysregulation of calpain, a prominent cysteine protease recognized toplay an important role in signal transduction, cell migration, andregulation of apoptosis, has been implicated in tissue damage followingischemic stroke. We have discovered, isolated, and/or made inhibitors ofcalpain which are specific for calpain.

We developed our peptidomimetic inhibitors of calpain by rational andsystematic chemical modifications of the amide bond and/or side chainfunctionalities of permutations of SEQ ID NO: 2, followed bycalpain-inhibitory kinetic measurements of the designed molecules invitro as well as an assessment of their ability to improve neurologicalrecovery in vivo after focal cerebral ischemia in rats. The structuralmodifications performed to create the peptidomimetics included, withoutlimitation, amino acid replacements by chemical mutagenesis, amide bondmimics, methylene group insertion between the α-carbon and the carbonylfunctionalities of individual amino acids, replacement of natural aminoacid side chains with non-natural moieties, and amino acid side chaincyclizations. Calpain-inhibitory activity of the peptidomimetics wasmeasured by steady-state and stopped-flow enzyme inhibition kineticstudies using purified calpain and natural protein or syntheticfluorogenic peptide substrates. The effect of the peptidomimetics oncerebral infarction, cerebral endothelial function and neurologicalfunctional recovery after ischemic stroke was assessed using a rat modelof 2 hour middle cerebral artery (“MCAO”) ischemia.

Based on our studies, we selected and further tested SEQ ID NO: 5 forits cerebroprotective capacity after 2 hour middle cerebral artery(“MCAO”) ischemia in the rat. Two hour MCAO by the suture method wasinduced in 21 male Wistar rats. Following reperfusion, intravenoussaline (n=9), SEQ ID NO: 5 (3 mg/kg; n=9), or SEQ ID NO: 5-βAla (3mg/kg; an inactive form of SEQ ID NO: 5, B27 β-Ala¹¹ in figures, n=9)was administered for 4 hours. Animals survived 7 days and neurologicaltesting (score 0-3, 3 being the most severe) was performed. Aftersacrifice the brain samples were formalin fixed and infarct volumes werecalculated using standard computerized image analysis. Independentt-test was used for statistical analysis.

All animals survived 7 days post MCA occlusion and treatment. As shownin FIGS. 1 and 2 (in all Figures herein, “B27-HYD” refers to SEQ ID NO:5), the group receiving SEQ ID NO: 5 had significantly reduced infarctvolume compared to the saline and a less active Leu¹¹ to βAla¹¹ versionof SEQ ID NO: 5, Asp Pro Met Ser Ser Thr Tyr Ile Glu Glu βAla Gly LysArg Glu Val Thr Ile Pro Pro Lys Tyr Val Ala Leu Leu Pro Ala Val Leu LeuAla Leu Leu Ala Pro (SEQ ID NO: 13), SEQ ID NO: 5 with β-Ala¹¹ treatedrats (24.9±3.8 vs 38.4±2.1 and 33.8±4.2; mean±SEM, p<0.05). As shown inFIG. 2, neurological scores, derived from behavioral and observationaltests of control and treated animals, were significantly improved at 7days in the SEQ ID NO: 5 treated group compared to the saline and SEQ IDNO: 13 (β-Ala¹¹ treated) groups (1.00±0.17 vs 1.78±0.22 and 1.44±0.18,mean±SEM, p<0.05).

We discovered that our selective calpain inhibitors arecerebroprotective after transient MCA occlusion in the rat. Oneadvantage of studying the mechanism of cerebral ischemia with SEQ ID NO:5 is its ability to selectively inhibit calpain, compared to othernon-calpastatin derived compounds. Selectively blocking calpain aftercerebral ischemia will enable further understanding of calpain'sspecific role in post-ischemic neuronal damage as well as increasingtherapeutic options.

Example 2

Calpastatin, a naturally occurring protein, is the only inhibitor thatis specific for calpain, and it is generally accepted that theinteraction of calpastatin with calpain is the most relevant mechanismresponsible for the regulation of Ca²⁺-inducted proteolysis. We haveidentified two “hot spots” in the peptide of SEQ ID NO: 2 that containthe residues critical for the potent and specific inhibition of calpain.To the best of our knowledge, none of the studies reported to date onthe pharmacologic inhibition of calpain has benefited from the absolutespecificity of calpastatin to delineate calpain's contribution to celldeath and neurological dysfunction following ischemic stroke. Thus, weevaluated SEQ ID NO: 5, one embodiment of a novel BBB-permeant analogueof SEQ ID NO: 2, for its effect on cerebral infarction and neurologicalfunctional recovery after focal cerebral ischemia in rats.

Materials and Methods

Animal Model.

Following an institutionally approved animal care protocol, male Wistarrats (270-290 g) were obtained from Charles River Breeding Co.(Wilmington, Mass., USA). Two-hour middle cerebral artery occlusion(MCAO) by the intravascular suture method, drug infusion, measurement ofinfarct volume and neurological deficits were performed as detailedpreviously.

SEQ ID NO: 5 and its less active Leu¹¹ to βAla¹¹ mutant SEQ ID NO: 13were prepared by FMOC-chemistry solid phase peptide synthesis andcharacterized for calpain inhibitory activity. SEQ ID NO: 5 (50 μMsolution, i.v. infusion at 15 μl/min for 4 h; 3 mg/kg), SEQ ID NO: 13 orvehicle (1% DMSO in saline) was administered to rats 97 (n=6-9)immediately after reperfusion following 2 h of MCAO. Sham-operatedanimals had surgery but no MCA occlusion. Animals to be studied forneurological score and infarct volume survived 7 days.

Brain protein extraction and Western blot analysis.

Following MCAO and drug infusion, the animals were sacrificed after 24or 48 h of survival. At that time, the brains were extracted andseparated into right and left, cortical and subcortical regions,snap-frozen in liquid nitrogen and stored at −80° C. until used. ForWestern blot analysis, the brain samples were pulverized to a finepowder with a small mortar and pestle set over dry ice. The pulverizedbrain tissue powder was homogenized in a solution of 0.25 M sucrose, 25mM 2-(N-morpholino)-ethanesulfonic acid, 1 mM EDTA, pH 6.5, containing0.025 mM E-64, 2.0 mM AEBSF, 0.5 mM PMSF, 0.02 mM leupeptin, 0.05 mMpepstatin, and 0.001 mM aprotinin (homogenization buffer) at 4° C. in aPotter-Elvehjem homogenizer. The homogenates were then centrifuged at 4°C. for 20 min at 16,000 g. Aliquots of the supernatant were stored at−80° C. until used for experiments. Protein concentrations weredetermined using micro BCA Protein Assay kit (Pierce, Rockford, Ill.).SDS-PAGE was carried out according to Laemmli and immunoblots wereperformed according to the technique of Towbin et al. Mouse monoclonalanti-all-spectrin antibody (MAB 1622, 1:1,000) and horse radishperoxidase (HRP)-conjugated goat anti-mouse IgG (AP124P, 1:10,000) werefrom Chemicon (Temecula, Calif.). Labeled proteins were detected withthe enhanced chemiluminescence method according to the manufacturer'sinstructions (GE Healthcare Biosciences, Piscataway, N.J., USA). Bandswere quantified by densitometry using Gel-Doc 2000 (Bio-Rad, Richmond,Va.) and the free open source software ImageJ.

Results:

Infarct Volume.

From the H&E sections, the ischemic core area with diffuse pallor of theeosinophilic background and the border area with vacuolation orsponginess and neutrophils can be identified under the microscope. Ascan be seen from the representative H&E stained serial coronal sectionsA through G in FIG. 3, the brain region mostly affected by the ischemiais the cortex in the MCA distribution (black arrows). The subcorticalregion in the core slices of C and D is also affected (white arrows).The ischemic damage (i.e. cell death, loss of tissue, orencephalomalacia) is less in the SEQ ID NO: 5-treated animals comparedto the vehicle-treated controls. By comparison, the contralateral sideappeared undamaged by both gross inspection and in histologicalsections. As shown in Table 1 and FIG. 1, SEQ ID NO: 5 significantlyreduced infarct volume compared to SEQ ID NO: 13-treated (24.9±3.8% vs.33.8±4.2%, percent hemispheric infarct volume±SE; ^(#)P<0.05,independent t-test; n=9 per group) and vehicle-treated (24.9±3.8% vs.38.4±2.1%, percent hemispheric infarct volume±SE; *P<0.05, independentt-test; n=9 per group) controls, respectively.

TABLE 1 Q1 Summary of data for infarct volume and neurologicalfunctional outcome. Lesion Volume Neurological Score Group % ofHemisphere 0 Day 1 Day 4 Days 7 Days Saline (n = 9) 38.4 ± 2.1 2.89 ±0.11 2.33 ± 0.24 1.89 ± 0.24 1.78 ± 0.22 SEQ ID NO: 5 (n = 9)  24.9 ±3.8*^(,#) 2.89 ± 0.11 2.33 ± 0.24 1.44 ± 0.18  1.00 ± 0.17*^(,#) SEQ IDNO: 13 (n = 9) 33.8 ± 4.2 2.89 ± 0.11 2.11 ± 0.20 1.56 ± 0.18 1.44 ±0.18 Values are mean ± SE. *P < 0.05 SEQ ID NO: 5 vs. vehicle. ^(#)P <0.05 SEQ ID NO: 5 vs. SEQ ID NO. 13

Neurological Score.

Neurological scores have been previously defined as follows: noobservable deficit=0, forelimb flexion=1, decreased resistance tolateral push with forelimb flexion=2, same behavior as 2 pluscircling=3. The behavioral data indicate that the neurological scoresslightly improved in all the experimental groups during the first 4 dayspost-injury (Table 1 and FIG. 2). However, the difference in performancebetween the SEQ ID NO: 5-treated animals and the vehicle controls(1.00±0.17 vs. 1.78±0.22, *P<0.05, n=9 per group; independent t-test)and the SEQ ID NO: 13-treated group (1.00±0.17 vs. 1.44±0.18,^(#)P<0.05, n=9 per group; independent t-test) was statisticallysignificant at day 7 post-injury.

αII-Spectrin Breakdown.

Pathologic activity of calpain and in vivo efficacy of SEQ ID NO: 5 wereassessed by Western blot analysis of all-spectrin breakdown products(SBDPs) generated in the rat brain following MCAO. FIG. 4 shows dataindicating that SEQ ID NO: 5 blocks calpain-mediated cleavage ofαII-spectrin in rat brain following MCAO: (A-D) Representativeimmunoblots of αII-spectrin in ipsilateral cortical (A,C) andsubcortical (B,D) regions of sham-operated, ischemic vehicle-treated,and ischemic SEQ ID NO: 5-treated animals at 24 h (A,B) and 48 h (C,D)following MCAO. Intact αII spectrin (280 kDa), SBDP150, SBDP145, SBDP120and SBDP110 are demonstrated. (E) Densitometric analysis ofSBDP150/SBDP145 levels in ipsilateral cortex and subcortex at 24 and 48h following MCAO.

As can be seen in FIG. 4, brain samples from sham-operated animalspresent mainly the intact αII-spectrin (280-kDa) while thestroke-injured vehicle-treated animals (vehicle controls) had elevatedlevels of 150- and 145-kDa SBDPs (SBDP150 and SBDP145) in the corticaland subcortical regions of the ipsilateral hemisphere in the first 24 hpost-injury (panels A and B). SEQ ID NO: 5 treatment blocked SBDP150 andSBDP145 formation to basal levels at 24 h post-injury (panel A, B, andE). In the vehicle control animals, SBDP145 levels continued to increasein the brain, becoming the predominant all-spectrin cleavage product at48 h post-injury (see panels C and D). It is interesting to note thatadditional SBDPs of 120 (SBDP120) and 110 kDa (SBDP110), which were notpresent in the first 24 h post-injury, were generated by 48 hpost-injury. SEQ ID NO: 5 treatment immediately after MCAO/reperfusionsignificantly reduced SBDP145 levels in the cortical and subcorticalregions of the brain at 48 h post-injury (*P<0.05 vs. vehicle; n=6; 180panel C, D, and E).

We have developed and used novel calpain inhibitors that exploitcalpastatin's absolute specificity for calpain and a BBB-permeable drugdelivery strategy and shows that postischemic inhibition of calpainsignificantly reduces infarct volume and improves neurologicalfunctional recovery after focal cerebral ischemia. In some embodiments,without limitation, a novel calpain inhibitor SEQ ID NO: 5 is comprisedof a signal sequence-derived membrane translocation peptide motifidentified through amino acids 23-36 of SEQ ID NO: 5 (VALLP AVLLA LLAP),conjugated to the C-terminus of human calpastatin subdomain B. SEQ IDNO: 5 is a potent, water-soluble, and specific inhibitor of calpain thatis non-toxic to cultured cells.

Investigation of the pathobiology of cerebral ischemia indicates thatthe initial ischemic insult induces massive release of glutamate fromdamaged synapses which leads to activation of glutamatereceptor-associated and voltage-dependent calcium channels. Suchactivation induces influx of calciumions into the neuron and release ofcalcium ions from intracellular stores. Loss of intracellular calciumhomeostasis contributes to cell death by activating various enzymes,including proteases, kinases, phosphatases, and phospholipases. Integralto the mechanism of calcium-mediated brain injury is the pathologicactivation of calpains. Under normal physiological conditions, calpainexists at very low activity in cells and is proposed to participate inthe turnover of cytoskeletal proteins and the regulation of kinases,transcription factors, and receptors. However, pathologic calpainactivation results in proteolytic destruction of many cellular proteinsincluding receptor proteins, calmodulin binding proteins, signaltransduction enzymes, transcription factors, and cytoskeletal proteins.Furthermore, uncontrolled calpain activity prevents increased expressionof several key proteins, including growth-associated protein-43(GAP-43), synaptophysin, and collapsin receptor mediator proteins(CRMPs), that play a major role in regeneration and neuroplasticityafter ischemic and traumatic brain injury. Thus, calpains can contributeto the pathogenesis of ischemic brain injury via multiple molecular andcellular pathways. Thus, selective inhibition of calpains may lead toboth cerebroprotective effects and an enhancement of neuronalplasticity/repair mechanisms.

We assessed functional outcome as an endpoint of our evaluation by themethod of Bederson et al. Treatment with SEQ ID NO: 5 immediately after2-h MCAO reduced neurological deficits by 24% and 44% on day 4 and day7, respectively, compared to vehicle-treated stroke controls. Infarctvolume was a secondary outcome and was measured at day 7 after stroke.The extent of cerebral infarction was reduced by 35% in the SEQ ID NO:5-treated animals compared to vehicle-treated animals Due to thepredictable location of areas of infarction and consistent production ofneurological deficits in the MCAO model used in this study, the effectsof SEQ ID NO: 5 on neurological outcome and size of infarction producedby focal cerebral ischemia could be reliably studied. It must be noted,however, that, infarction volumes could correlate poorly with functionaloutcome because small lesions in cortical locations can produce majorfunctional deficits. Conversely, large lesions in relatively silentareas cause little detectable function loss. Reduction of neurologicaldeficits after stroke has been attributed to synaptic and functionalreorganization in the cerebral cortex and in subcortical structuresafter ischemic injuries. Since pathologic calpain activity can result indisruption of axonal transport and structural collapse as well as a netdecrease in the level of proteins involved in the neuronal plasticitythat occurs after brain injury, our data indicate that SEQ ID NO: 5 mayeffectively treat an array of calpain-mediated physiological changesassociated with stroke to improve functional outcome.

The most well-studied calpain target is αII-spectrin, a 280-kDa neuronalprotein that localizes to axons and functions in cortical cytoskeletonmatrix support. αII-spectrin is proteolyzed by calpain to generate 150-and 145-kDa spectrin breakdown products (SBDP150 and SBDP145) or bycaspase-3 to generate 150- and 120-kDa fragments (SBDP150i and SBDP120)following ischemic and traumatic brain injury. Calpain-generated SBDP145is a signature biomarker for neuronal necrotic/oncotic cell death afterischemic and traumatic brain injury while caspase-3-generated SBDP120 isa signature biomarker for neuronal apoptosis. Recently, the clinicalsignificance of calpain and caspase-3 specific SBDPs as biomarkers foracute brain injury was demonstrated in humans. Our data confirm thatnecrotic/oncotic and apoptotic cell death mechanisms overlap but appearto be activated at distinct time patterns after MCAO. As shown in FIGS.4A and B, the extent of SBDP145 formation was similar in both corticaland subcortical regions of the ischemic hemisphere at 24 h post-injury.Interestingly, the SBDP145 levels increased about 3- and 2-fold in thecortex and subcortex, respectively, by 48 h after MCAO, suggestingcontinued calpain-mediated proteolysis of all-spectrin in the ischemichemisphere probably due to a new surge in intracellular Ca²⁺ ([Ca²⁺]i)levels in this region of the brain that resulted in an additionalincrease in calpain activation between 24 and 48 h following the initialischemic injury. While acute postischemic administration of SEQ ID NO: 5completely blocked SBDP145 accumulation at 24 h post-injury, thetreatment significantly reduced SBDP145 levels in the cortex and thesubcortex at 48 h. In addition to its use in assessing neuronalstructural degradation and evaluating possible mechanisms involved inthe evolving brain damage after stroke, we have demonstrated thepotential utility of SBDP145 biomarker as an adjunct in guidingtherapeutic decision making during the development ofcalpain-target-based neuroprotective strategies.

To date, preclinical studies reported on anti-calpain treatmentstrategies for stroke have used inhibitor designs based ondiortripeptide and peptidomimetic address labels modified with achemical warhead. Even though these small molecule synthetic inhibitorsare effective in inhibiting calpain, they do, to varying degrees, reactwith other proteases implicated in the pathophysiology of stroke. Itcould be argued that single-drug therapy with a broad-spectrum inhibitorthat blocks several proteases involved in the progression of ischemicbrain injury would be more effective than treatment with a highlyselective inhibitor that targets one particular protease. However, thelack of specificity of a protease inhibitor often raises other issuesrelated to tissue toxicity. Furthermore, the onset, duration, and extent(amount) of abnormal protease activity generated after the initialischemic event are likely to be different for each protease target. Ourfindings indicate that a protease-targets-based combination therapyaimed at selective inhibition of unique steps of protease-mediated braininjury would result in synergistic brain tissue protection andimprovement of functional outcome after stroke.

Example 3

We used SEQ ID NO: 5 to investigate the role of calpain in neurovasculardysfunction after stroke.

Two-hour middle cerebral artery occlusion (MCAO) was used to induceischemic stroke in Male Wistar rats. SEQ ID NO: 5 was givenintravenously to rats immediately after reperfusion following 2 h ofMCAO, while control animals had infusion of vehicle solution (n=6-9).Animals to be studied for neurological score and infarct volume survived7 days, and neurological testing (mNSS and corner turn) were performedat 1, 4, and 7 days after MCAO. In another set of experiments, theanimals were sacrificed at 48 h, and histology was performed for BBBintegrity (albumin and fibrin leakage from the cerebral microvasculatureinto brain parenchyma). Phosphorylation of endothelial nitric oxidesynthase (eNOS) was analyzed by Western blotting with specificantibodies raised against synthetic phosphorylated peptides representingknown eNOS phosphorylation sites.

Results:

Postischemic administration of SEQ ID NO: 5 reduced infarct volume andneurological deficits by 35% and 44%, respectively, compared tountreated animals (p<0.05 for each). BBB integrity as assessed byalbumin and fibrin extravasation showed significant improvement afterSEQ ID NO: 5 treatment compared to controls (p<0.05) at 48 h.Endothelial nitric oxide synthase (eNOS) threonine-495 phosphorylationwhich was found to be highly upregulated (˜3-fold) in the brain afterstroke was significantly reduced by treatment with SEQ ID NO: 5. Thus,we discovered unexpectedly that treatment of transient cerebral ischemiawith a specific calpastatin-based inhibitor of calpain hasneuroprotective and functional recovery associated with regulation ofeNOS and vasoprotection, as can be seen from its beneficial effects onBBB integrity.

FIG. 5 shows data indicating that SEQ ID NO: 5 reduces rat serum albumin(RSA) leakage into the ischemic brain parenchyma. Shown arerepresentative immunoblots of RSA levels in the ipsilateral cortical (A)and subcortical (B) regions of sham-operated, ischemic vehicle-treated,and ischemic. C, Densitometric analysis of RSA levels in ipsilateralcortex and subcortex (*p<0.05 vs vehicle; n=6) at 24 h following MCAO.

FIG. 6 shows data indicating that SEQ ID NO: 5 treatment decreasesfibrin deposition outside the cerebral microvasculature. Representativefluorescent micrographs of fibrin deposition outside the cerebralmicrovasculature of untreated (MCAO, panel A) and treated (+SEQ ID NO:5, panel B) rats subjected to 2 hour MCA occlusion with reperfusion and48 hour survival are shown. A schematic representation of a coronalbrain section showing eight fields selected from the ischemic boundaryzone (IBZ) for quantitative measurements of immunochemical staining forfibrin in the cortex and striatum is shown (panel C). The quantitativedata confirms that SEQ ID NO: 5 treatment of stroke significantlydecreases BBB leakage compared to control MCAO rats (panel D).

FIG. 7 shows data indicating that SEQ ID NO: 5 modulates eNOSphosphorylation state: A-B: Both upper and lower bands arephosphorylated at eNOS-Ser633. C: Graphical representation of the ratioof upper:lower phospho-eNOS protein band intensities (n=6rats/experimental group).

FIG. 8 shows data indicating that post-injury treatment with SEQ ID NO:5 induces eNOS-Thr495 dephosphorylation

Stroke is a leading cause of death and long-term disability without anydefinitive treatments that have been demonstrated to improve functionaloutcomes. Accumulated evidence implicates calpains in the pathogenesisof ischemic brain injury via multiple molecular and cellular pathways.In vivo inhibition of calpain using cysteine protease inhibitors (e.g.peptide aldehydes, alpha-keto esters and amides) diminishes the extentof neuronal damage following ischemia; however, the relativenon-specificity of these cysteine protease inhibitors precludes adefinitive role for calpain participation. Traditionally,calpain-mediated postischemic neurological dysfunction has beenattributed mainly to the enzyme's destructive proteolysis ofcytoskeletal proteins leading to necrotic and/or apoptotic cell death.However, calpains can also participate in neurovascular cell signalingevents via proteolytic activation of kinases and phosphatases. Nitricoxide (NO) serves as an endogenous vasodilator, platelet inhibitor,antioxidant, and regulator of vascular endothelium by sustaining itsanti-coagulant and anti-thrombogenic properties. Mice lacking theconstitutively expressed form of endothelial nitric oxide synthase(eNOS) exhibit larger cerebral infarctions and upregulation of eNOS withL-arginine and statins protects against stroke. More importantly, eNOSactivity is regulated through multiple and coordinated phosphorylationevents at various phosphorylation sites mediated by multiple kinases.The literature to date indicates that six specific sites ofphosphorylation have been identified in human eNOS at Tyr81, Ser114,Thr495, Ser615, Ser633, and Ser1177. Depending on the site,phosphorylation of eNOS could either activate the enzyme or attenuateenzyme activity. Phosphorylation of eNOS-Ser633 increases eNOS activityand appears particularly important for maintenance of NO synthesis afterinitial activation by Ca2+ flux and eNOS-Ser1177 phosphorylation. Incontrast, phosphorylation of eNOS-Thr495 in the Ca2+/calmodulin (CaM)binding domain is inhibitory. In this study, we benefited from theabsolute specificity of calpastatin to inhibit calpain pharmacologicallyand to investigate the mechanism of calpain in neurovascular dysfunctionafter stroke.

Our findings substantiate the involvement of calpain in postischemicneurological and cerebrovascular dysfunction and show a protectivemechanism of SEQ ID NO: 5. The protective mechanism is, in part, due topreservation of cerebral endothelial function. An interesting finding isthat calpain is involved in regulation of endothelial nitric oxidesynthase, a key enzyme involved in neurovascular function, throughphosphorylation events. SEQ ID NO: 5 as a novel BBB-permeant specificinhibitor of calpain provides a novel approach for the treatment ofstroke, as well as for investigating the mechanism(s) ofcalpain-mediated neurodegenerative disorders. Embodiments of theinvention may expand the therapeutic window for ischemic stroke andcould be applied to a large portion of the many patients who sufferstroke each year worldwide.

Thus, without limitation and without disclaimer of subject matter, someembodiments comprise novel compositions and methods to prevent, control,or alleviate mammalian injury, including without limitation, braindamage, through the selective application of novel calpain inhibitors.In accordance with some embodiments, without limitation, one may inhibitsuch trauma through the administration of one or more such inhibitorsfor a finite interval of time, thereby limiting the development of suchdamage.

In accordance with some embodiments, there is a high likelihood that theduration of drug therapy would be relatively brief and with a highprobability of success. Prophylactic administration of efficaciousamounts of the calpain inhibitors of some embodiments may greatly reducethe incidence of damage associated with many forms of neural trauma.

In accordance with some embodiments, a route of administration of thecalpain inhibitors of some embodiments in humans is by oraladministration. However, any appropriate routes of administering suchinhibitors known to those of ordinary skill in the art also compriseembodiments of the invention.

The calpain inhibitor(s) of some embodiments would be administered anddosed in accordance with good medical practice, taking into account theclinical condition of the individual patient, the site and method ofadministration, scheduling of administration, patient age, sex, bodyweight and other factors known to medical practitioners. The“pharmaceutically effective amount” for purposes herein is thusdetermined by such considerations as are known in the art. The amountmust be effective to achieve improvement, including but not limited to,decreased damage or injury, or improvement or elimination of symptomsand other indicators as are selected as appropriate measures by thoseskilled in the art.

In accordance with some embodiments, such calpain inhibitor(s) can beadministered in various ways. It can be administered alone or as anactive ingredient in combination with pharmaceutically acceptablecarriers, diluents, adjuvants and vehicles. The inhibitor(s) can beadministered orally, subcutaneously or parenterally includingintravenous, intraarterial, intramuscular, intraperitoneal, andintranasal administration as well as intrathecal and infusiontechniques, or by local administration or direct inoculation to the siteof disease or pathological condition. Implants of the compounds are alsouseful. The patient being treated is a warm-blooded animal and, inparticular, mammals including humans. The pharmaceutically acceptablecarriers, diluents, adjuvants and vehicles as well as implant carriersgenerally refer to inert, non-toxic solid or liquid fillers, diluents orencapsulating material not reacting with the active ingredients of theinvention.

Since the use of such the calpain inhibitors in accordance with someembodiments specifically targets the evolution and expression ofassociated pathologies, it is expected that the timing and duration oftreatment in humans will approximate those established for animalmodels. Similarly, the doses established for achieving desired effectsusing such compounds in animal models, or for other clinicalapplications, would be expected to be applicable in this context aswell. SEQ ID NO: 5 is water soluble and dissolved in PBS. We haveinfused SEQ ID NO: 5 at a concentration of 50 micromolar solution viafemoral vein for 4 hours after reperfusion of the middle cerebral arteryfor a total dose of 3 mg/kg. This dose was effective at improvingneurological recovery in this stroke model. When studying anothercysteine protease inhibitor (which blocks cathepsins B and L) in thisstroke model in a similar fashion, we found that 4 hour infusion ofconcentrations of 10, 50, and 250 micromolar were equally effective atreducing neurological impairment, whereas 2 micromolar solutions was noteffective. It is noted that humans are treated generally longer than theexperimental animals exemplified herein which treatment has a lengthproportional to the length of the disease process and drugeffectiveness. The doses may be single doses or multiple doses overperiods of time. The treatment generally has a length proportional tothe length of the disease process and drug effectiveness and the patientspecies being treated. Without limiting or disclaiming any embodiments,a therapeutically effective amount of the compound described hereinpreferably ranges from about 0.01 milligrams per kilogram of body weightper day (mg/kg/day) to about 100 mg/kg/day. Preferred amounts areexpected to vary from about 0.5 to about 30 mg/kg/day.

When administering the calpain inhibitor(s) of some embodimentsparenterally, it will generally be formulated in a unit dosageinjectable form (solution, suspension, emulsion). The pharmaceuticalformulations suitable for injection include sterile aqueous solutions ordispersions and sterile powders for reconstitution into sterileinjectable solutions or dispersions. The carrier can be a solvent ordispersing medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, liquid polyethylene glycol, and thelike), suitable mixtures thereof, and vegetable oils.

When necessary, proper fluidity can be maintained, for example, by theuse of a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Nonaqueous vehicles such a cottonseed oil, sesame oil, olive oil,soybean oil, corn oil, sunflower oil, or peanut oil and esters, such asisopropyl myristate, may also be used as solvent systems for suchcalpain inhibitor(s) compositions. Additionally, various additives whichenhance the stability, sterility, and isotonicity of the compositions,including antimicrobial preservatives, antioxidants, chelating agents,and buffers, can be added. Prevention of the action of microorganismscan be ensured by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, and the like. Inmany cases, it will be desirable to include isotonic agents, forexample, sugars, sodium chloride, and the like. Prolonged absorption ofthe injectable pharmaceutical form can be brought about by the use ofagents delaying absorption, for example, aluminum monostearate andgelatin. According to the present invention, however, any vehicle,diluent, or additive used would have to be compatible with the calpaininhibitor(s).

Sterile injectable solutions can be prepared by incorporating theinhibitor(s) utilized in practicing the present invention in therequired amount of the appropriate solvent with various of the otheringredients, as desired.

A pharmacological formulation of some embodiments may be administered tothe patient in an injectable formulation containing any compatiblecarrier, such as various vehicle, adjuvants, additives, and diluents; orthe inhibitor(s) utilized in some embodiments may be administeredparenterally to the patient in the form of slow-release subcutaneousimplants or targeted delivery systems such as monoclonal antibodies,vectored delivery, iontophoretic, polymer matrices, liposomes, andmicrospheres. Many other such implants, delivery systems, and modulesare well known to those skilled in the art.

In some embodiments, without limitation, the calpain inhibitor(s) ofsome embodiments may be administered initially by intravenous injectionto bring blood levels to a suitable level. The patient's levels are thenmaintained by an oral dosage form, although other forms ofadministration, dependent upon the patient's condition and as indicatedabove, can be used. The quantity to be administered and timing ofadministration may vary for the patient being treated.

This application may reference various publications by author, citation,and/or by patent number, including without limitation, articles,presentations, and United States patents. The disclosures of each ofthese references in their entireties are hereby incorporated byreference into this application.

While the present invention has been particularly shown and describedwith reference to the foregoing preferred and alternative embodiments,it should be understood by those skilled in the art that variousalternatives to the embodiments of the invention described herein may beemployed in practicing the invention without departing from the spiritand scope of the invention as defined in the following claims. It isintended that the following claims define the scope of the invention andthat the method and apparatus within the scope of these claims and theirequivalents be covered thereby. This description of the invention shouldbe understood to include all novel and non-obvious combinations ofelements described herein, and claims may be presented in this or alater application to any novel and non-obvious combination of theseelements. The foregoing embodiments are illustrative, and no singlefeature or element is essential to all possible combinations that may beclaimed in this or a later application. Where the claims recite “a” or“a first” element of the equivalent thereof, such claims should beunderstood to include incorporation of one or more such elements,neither requiring nor excluding two or more such elements.

Disclosed Nucleic Acid And Peptide Sequences

SEQ ID NO: 1: acc caa tgg cta cta cct acc taa gag gaa ttg ggt  aaa aga gaa gtc aca att cct cca aaa tat agg gaa  cta ttg gctSEQ ID NO: 2: Asp Pro Met Ser Ser Thr Tyr Ile Glu Glu Leu Gly  Lys Arg Glu Val Thr Ile Pro Pro Lys Tyr Arg Glu  Leu Leu AlaSEQ ID NO: 3: Asp Pro Met Ser Ser Thr Tyr Ile Glu Glu Leu Gly  Lys Arg Glu Val Thr Ile Pro Pro Lys Tyr Val Ala  Leu Leu ProSEQ ID NO: 4: Ala Val Leu Leu Ala Leu Leu Ala Pro SEQ ID NO: 5:Asp Pro Met Ser Ser Thr Tyr Ile Glu Glu Leu Gly  Lys Arg Glu Val Thr Ile Pro Pro Lys Tyr Val Ala Leu Leu Pro Ala Val Leu Leu Ala Leu Leu Ala Pro SEQ ID NO: 6:Asp Pro Met Ser Ser Thr Tyr Ile Glu Glu Leu Gly  Lys Arg Glu Val Thr Nva Pro Pro Lys Tyr Orn Glu  Leu Leu AlaSEQ ID NO: 7: Asp Pro Met Ser Ser Thr Tyr Ile Glu Glu Leu Gly  Lys Arg Glu Val Thr Nva Pro Pro Lys Tyr Arg Glu  Leu Leu AlaSEQ ID NO: 8: Asp Pro Met Ser Ser Thr Tyr Ile Glu Glu Leu Gly  Lys Arg Glu Val Thr Ile Pro Pro Lys Tyr Orn Glu  Leu Leu AlaSEQ ID NO: 9: Asp Pro Met Ser Ser Thr Tyr Ile Glu Glu Leu Gly  Lys Arg Glu Val Thr Nva Pro Pro Lys Tyr Val Ala  Leu Leu ProSEQ ID NO: 10: Asp Pro Met Ser Ser Thr Tyr Ile Glu Glu Leu Gly  Lys Arg Glu Val Thr Nva Pro Pro Lys Tyr Val Ala Leu Leu Pro Ala Val Leu Leu Ala Leu Leu Ala Pro SEQ ID NO: 11:gcc gcg gta gcg ctg ctc ccg gcg gtc ctg ctg gcc  ttg ctg gcg cccSEQ ID NO: 12: Ala Ala Val Ala Leu Leu Pro Ala Val Leu Leu Ala Leu Leu Ala Pro SEQ ID NO: 13:Asp Pro Met Ser Ser Thr Tyr Ile Glu Glu βAla Gly  Lys Arg Glu Val Thr Ile Pro Pro Lys Tyr Val Ala Leu Leu Pro Ala Val Leu Leu Ala Leu Leu Ala Pro

What is claimed is:
 1. A synthetic peptide comprising SEQ ID NO:
 5. 2. Acomposition comprising: the peptide of claim 1; and an additive selectedfrom the group consisting of a hydrophobic blood-brain-barrier or amembrane-permeant amino acid sequence linked by a polyethylene glycollinker to the N-terminus of said peptide, SEQ ID NO. 11, SEQ ID NO. 12,a nonaqueous vehicle, a solvent, a diluent, adjuvant, a chelating agent,a stability enhancing additive, antimicrobial preservatives,antioxidants, buffers, an antibacterial or an antifungal agents,parabens, chlorobutanol, phenol, sorbic acid, an isotonic agent, asugar, sodium chloride, a prolonged absorption agent, aluminummonostearate, a gelatin and combinations thereof.
 3. A method ofinhibiting neuronal damage in a subject following an ischemic event,comprising the steps of: providing the composition of claim 2, andadministering a pharmaceutically effective amount of said composition tothe subject.
 4. A drug for inhibiting neural damage in a subjectfollowing an ischemic event, the drug comprising: the peptide of claim1; and a pharmaceutically acceptable excipient or other additiveselected from the group consisting of a hydrophobic blood-brain-barrieror a membrane-permeant amino acid sequence linked by a polyethyleneglycol linker to the N-terminus of said peptide, SEQ ID NO. 11, SEQ IDNO. 12, a nonaqueous vehicle, a solvent, a diluent, adjuvant, achelating agent, a stability enhancing additive, antimicrobialpreservatives, antioxidants, buffers, an antibacterial or an antifungalagents, parabens, chlorobutanol, phenol, sorbic acid, an isotonic agent,a sugar, sodium chloride, a prolonged absorption agent, aluminummonostearate, a gelatin and combinations thereof.
 5. A compositioncomprising: one or more peptides of SEQ ID NOS: 3, 5, 6, 7, 8, 9, and10.; and a pharmaceutically acceptable excipient or other additiveselected from the group consisting of a hydrophobic blood-brain-barrieror a membrane-permeant amino acid sequence linked by a polyethyleneglycol linker to the N-terminus of said peptide, SEQ ID NO. 11, SEQ IDNO. 12, a nonaqueous vehicle, a solvent, a diluent, a chelating agent, astability enhancing additive, antimicrobial preservatives, antioxidants,buffers, an antibacterial or an antifungal agents, parabens,chlorobutanol, phenol, sorbic acid, an isotonic agent, a sugar, sodiumchloride, a prolonged absorption agent, aluminum monostearate, a gelatinand combinations thereof.
 6. A method of inhibiting neuronal damage in asubject following an ischemic event, comprising the steps of: providingthe composition of claim 5, and administering a pharmaceuticallyeffective amount of said composition to the subject.
 7. A drug forinhibiting neural damage in a subject following an ischemic event, thedrug comprising: one or more peptide of SEQ ID NOS: 3, 5, 6, 7, 8, 9, or10; and a pharmaceutically acceptable excipient or other additiveselected from the group consisting of a hydrophobic blood-brain-barrieror a membrane-permeant amino acid sequence linked by a polyethyleneglycol linker to the N-terminus of said peptide, SEQ ID NO. 11, SEQ IDNO. 12, a nonaqueous vehicle, a solvent, a diluent, a chelating agent, astability enhancing additive, antimicrobial preservatives, antioxidants,buffers, an antibacterial or an antifungal agents, parabens,chlorobutanol, phenol, sorbic acid, an isotonic agent, a sugar, sodiumchloride, a prolonged absorption agent, aluminum monostearate, a gelatinand combinations thereof.
 8. A synthetic peptide comprising SEQ ID NO: 2or SEQ ID NO: 5 wherein the peptidic bond(s) of one or more of thefollowing amino acid pairs in the sequence is replaced with anon-peptidic reduced amide bond: Ser4-Ser5, Tyr6-Ile7, Lys13-Arg14,Glu15-Val16, Lys21-Tyr22, Tyr22-Arg23.
 9. A method of inhibitingneuronal damage in a subject following an ischemic event, comprising thesteps of: providing a composition comprised of the synthetic peptide ofclaim 8, and administering a pharmaceutically effective amount of saidcomposition to the subject.
 10. A drug for inhibiting neural damage in asubject following an ischemic event, the drug comprising: the syntheticpeptide of claim 8; and a pharmaceutically acceptable excipient or otheradditive selected from the group consisting of a hydrophobicblood-brain-barrier or a membrane-permeant amino acid sequence linked bya polyethylene glycol linker to the N-terminus of said peptide, SEQ IDNO. 11, SEQ ID NO. 12, a nonaqueous vehicle, a solvent, a diluent, achelating agent, a stability enhancing additive, antimicrobialpreservatives, antioxidants, buffers, an antibacterial or an antifungalagents, parabens, chlorobutanol, phenol, sorbic acid, an isotonic agent,a sugar, sodium chloride, a prolonged absorption agent, aluminummonostearate, a gelatin and combinations thereof.
 11. A syntheticpeptide comprising SEQ ID NO: 2, wherein there is side-chain cyclizationbetween Lys21 and Glu24.
 12. A method of inhibiting neuronal damage in asubject following an ischemic event, comprising the steps of: providinga composition comprised of the synthetic peptide of claim 11, andadministering a pharmaceutically effective amount of said composition tothe mammal.
 13. A drug for inhibiting neural damage in a subjectfollowing an ischemic event, the drug comprising: the synthetic peptideof claim 11; and a pharmaceutically acceptable excipient or otheradditive selected from the group consisting of a hydrophobicblood-brain-barrier or a membrane-permeant amino acid sequence linked bya polyethylene glycol linker to the N-terminus of said peptide, SEQ IDNO. 11, SEQ ID NO. 12, a nonaqueous vehicle, a solvent, a diluent, achelating agent, a stability enhancing additive, antimicrobialpreservatives, antioxidants, buffers, an antibacterial or an antifungalagents, parabens, chlorobutanol, phenol, sorbic acid, an isotonic agent,a sugar, sodium chloride, a prolonged absorption agent, aluminummonostearate, a gelatin and combinations thereof.